Oxidase Test – Principle, Procedure and Result

Catalase and Oxidase tests are performed to check whether the given microorganism is aerobic or anaerobic.

Oxidase Test –

Oxidase test is performed to detect the presence of intracellular cytochrome oxidase system in the test organism. The cytochrome oxidase is component of Electron Transport Chain (ETC). Electron transport chain the last step of cellular respiration. It is involved in the oxidation of the NADH and FADH2. While oxidation of NADH and FADH2, they donate electron to NADH-CoQ reductase, and it is further transferred to succinate dehydrogenase, cytochrome bc1, cytochrome oxidase complex and finally the electron is accepted by oxygen. The transfer of electrons causes pumping of hydrogen ion in inter-membranous space. The difference in gradient is exploit for ATP synthesis. This is majorly done by cytochrome oxidase complex. This results in difference in potential gradient across of the membrane. The cytochrome oxidase converts the reactive species like H⁺ and dioxygen (O₂) into water. Hence, cytochrome oxidase is involved in obtaining energy and converting reactive species to safe one.

Principle –

The presence of cytochrome oxidase can be tested using N, N, N’, N’-tetramethyl-p-phenylenediamine dihydrochloride (Kovacs)reagent. The reagent acts like an artificial electron acceptor for cytochrome oxidase in place of oxygen. If given microbe contains cytochrome oxidase, the reagent get oxidised, it forms indophenol blue and appears blue in color. Whereas, the colorless indicates the absence of cytochrome oxidase.

Procedure –

Filter Paper Method –

1. Soak a piece of whatman filter paper in 1% Kovacs oxidase reagent and allow it to dry.
2. Pick some bacterial colonies with the help of toothpick and place of Kovacs treated filter paper.
3. Rub the colonies on the surface of the paper.
4. Observe the appearance of blue color.

Result –

If blue color appears in 5 to 10 seconds, then the microorganisms are oxidase positive.

If blue color appears within 60 to 90 seconds, then it is called delayed positive oxidase test.

If blue color appears after two minutes then it is false positive test.

If there is no blue color development then it is negative test.

References –

https://asm.org/getattachment/00ce8639-8e76-4acb-8591-0f7b22a347c6/oxidase-test-protocol-3229.pdf

https://chem.libretexts.org/Courses/Saint_Marys

https://learn.chm.msu.edu/vibl/content/catalase.html